WO2020133643A1 - 一种人Vγ9Vδ2T细胞扩增培养方法与培养基 - Google Patents

一种人Vγ9Vδ2T细胞扩增培养方法与培养基 Download PDF

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WO2020133643A1
WO2020133643A1 PCT/CN2019/075491 CN2019075491W WO2020133643A1 WO 2020133643 A1 WO2020133643 A1 WO 2020133643A1 CN 2019075491 W CN2019075491 W CN 2019075491W WO 2020133643 A1 WO2020133643 A1 WO 2020133643A1
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cancer
cells
vitamin
medium
cell
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English (en)
French (fr)
Chinese (zh)
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尹芝南
吴扬哲
徐艳
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广东暨德康民生物科技有限责任公司
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Priority to EP19905880.1A priority Critical patent/EP3904506A4/en
Priority to JP2021536329A priority patent/JP7295243B2/ja
Priority to US17/418,103 priority patent/US20210332328A1/en
Priority to GB2109020.4A priority patent/GB2595372B/en
Priority to KR1020217018384A priority patent/KR20210091782A/ko
Priority to AU2019411781A priority patent/AU2019411781B2/en
Publication of WO2020133643A1 publication Critical patent/WO2020133643A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present disclosure relates to the technical field of cell culture, in particular to a method and medium for expanding human V ⁇ 9V ⁇ 2T cells.
  • Immune cell therapy has become a new important medical method for the treatment of refractory diseases (such as malignant tumors) in the medical field worldwide, such as the use of CAR-T cells to treat B-cell lymphoma.
  • refractory diseases such as malignant tumors
  • CAR-T cells to treat B-cell lymphoma.
  • obtaining immune cells with stable and reliable quality and excellent functions is crucial for obtaining good clinical treatment effects.
  • Human V ⁇ 9V ⁇ 2 T cells are a subset of T cells with anti-tumor and anti-infection functions that exist only in primates and humans. Human V ⁇ 9V ⁇ 2 T cells can be used for immune cell therapy or immune function reconstruction in people with tumors, refractory infectious diseases and immune imbalance diseases.
  • the first type of existing amplification technology is the addition of monoclonal antibodies to IL-2 and TCR to the culture medium of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the main disadvantage of this method is that both types of ⁇ T cells (V ⁇ 1 cell subset and V ⁇ 2 cell subset) in peripheral blood are expanded, and V ⁇ 1 cell subsets have immunosuppressive function, so they are not suitable for immune cell therapy.
  • the second type of existing amplification technology is to add IL-2 and phosphate small molecule compounds such as zoledronic acid to the culture medium of PBMCs.
  • This method can obtain ⁇ T cells of V ⁇ 2 subpopulation with higher purity, which can be used for immune cell therapy.
  • This is a common method for expanding human peripheral blood human V ⁇ 9V ⁇ 2 T cells at home and abroad.
  • the shortcoming of this method is that the cycle of cell culture expansion is about 14 days, the survival time of the expanded human V ⁇ 9V ⁇ 2 T cells is short (it can continue to survive for about 7 days), and the cell is not strong against apoptosis, which is The cell's killing ability is not strong.
  • the objectives of the present disclosure include, for example, to provide a method for expanding human V ⁇ 9V ⁇ 2T cells, which can improve the proliferation efficiency and cell purity of human V ⁇ 9V ⁇ 2 T cells, and the cultured human V ⁇ 9V ⁇ 2T cells have strong tumor killing ability and inhibitory ability, while resisting apoptosis The ability is stronger and the cells survive longer.
  • the purpose of the present disclosure also includes, for example, providing a human V ⁇ 9V ⁇ 2T cell culture medium, which is used to culture human V ⁇ 9V ⁇ 2T cells, which can improve the proliferation efficiency and cell purity of human V ⁇ 9V ⁇ 2 T cells, and the cultured human V ⁇ 9V ⁇ 2T cells have strong The tumor killing ability and inhibiting ability, while the anti-apoptosis ability is stronger, and the cell survival time is longer.
  • the purpose of the present disclosure also includes, for example, providing a human V ⁇ 9V ⁇ 2T cell stimulation expansion medium capable of selectively expanding human V ⁇ 9V ⁇ 2T cells from peripheral blood mononuclear cells, and the amplified human V ⁇ 9V ⁇ 2T cells have high purity and killing ability With stronger anti-apoptosis ability.
  • the present disclosure provides a T cell expansion method, including:
  • Step A using a first medium capable of stimulating T cells to culture a composition containing T cells to stimulate the T cells;
  • Step B Use the second medium to culture the stimulated T cells; wherein the second medium includes interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the T cells include V ⁇ 9V ⁇ 2 T cells.
  • the first medium includes interleukin-2 and phosphonic acid compounds.
  • the first medium includes interleukin-2, phosphonic acid compounds, interleukin-15, and vitamin C or vitamin C derivatives.
  • the vitamin C derivative is selected from vitamin C ethyl ether, vitamin C palmitate, vitamin C glucoside, vitamin C phosphate magnesium and vitamin C phosphate sodium.
  • the concentration of interleukin-15 in the second medium is 1-1000 ng/ml.
  • the concentration of vitamin C or vitamin C derivative in the second medium is 10 ⁇ M-800 mM.
  • the concentration of interleukin-2 in the second medium is 1-1000 ng/ml.
  • the T cell-containing composition includes peripheral blood mononuclear cells extracted from human peripheral blood.
  • the cultivation time in step A is 60-100 hours.
  • the phosphonic acid compounds include bisphosphonic acid compounds, preferably selected from zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid Acid, risedronic acid, minophosphoric acid, and combinations thereof, such as zoledronic acid.
  • the second medium includes basal medium, interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the basal medium is selected from the group consisting of RPMI-1640 medium, D-MEM, MEM, RPMI, Opti-MEM, and combinations thereof, such as RPMI-1640 medium.
  • the present disclosure also provides a culture medium, including basal medium, interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the concentration of interleukin-2 in the medium is 1-1000 ng/ml
  • the concentration of interleukin-15 is 1-1000 ng/ml
  • the concentration of vitamin C or vitamin C derivative is 10 ⁇ M- 800mM.
  • the present disclosure also provides a pharmaceutical composition, including T cells expanded using the amplification method herein and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a pharmaceutical composition, including V ⁇ 9V ⁇ 2T cells amplified using the amplification method herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell suspension.
  • the present disclosure also provides the use of T cells amplified by the amplification method of the present disclosure or the pharmaceutical composition of the present disclosure for the preparation of a medicament for inhibiting, preventing or treating infectious diseases, autoimmune diseases or malignant diseases .
  • the malignant disease is cancer.
  • the malignant disease is lung cancer.
  • the present disclosure also provides a method for inhibiting, preventing or treating infectious diseases, autoimmune diseases or malignant diseases, including:
  • peripheral blood mononuclear cells are expanded by the expansion method of the present disclosure.
  • the present disclosure also provides T cells expanded according to the expansion method herein or the pharmaceutical composition herein for use in inhibiting, preventing or treating infectious diseases, autoimmune diseases or malignant diseases.
  • the present disclosure also provides the use of the medium herein for expanding T cells.
  • the T cells are V ⁇ 9V ⁇ 2 T cells.
  • the present disclosure provides a method for expanding human V ⁇ 9V ⁇ 2T cells, including:
  • Step 1 Extract peripheral blood mononuclear cells from human peripheral blood
  • Step 2 Provide a first culture medium, and use the first culture medium to resuspend the peripheral blood mononuclear cells to obtain a cell suspension;
  • the first medium includes a second medium and phosphonic acid compounds;
  • the second medium includes a basal medium and interleukin-2, interleukin-15 and vitamin C or vitamin C added to the basal medium derivative;
  • Step 3 inoculate the cell suspension into a cell culture vessel and cultivate for 60-100 hours;
  • step 4 the second culture medium is used for liquid exchange, and subsequent culture processes are all performed by using the second culture medium.
  • the basal medium is RPMI-1640 medium
  • the phosphonic acid compounds include zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, a One or more of lendronic acid, risedronic acid, minophosphoric acid.
  • the cell density of the cell suspension is 3-4 ⁇ 106 cells/ml.
  • the concentration of the phosphonic acid compound in the first medium is 1-1000 ⁇ M
  • the concentration of interleukin-2 is 1-1000 ng/ml
  • the concentration of interleukin-15 is 1-1000 ng/ml
  • the concentration of vitamin C or vitamin C derivative is 10 ⁇ M -800mM.
  • the cell culture container is a 24-well plate, a 48-well plate, or a 96-well plate; in step 3, the cell suspension is inoculated into a cell culture container and cultured for 72 hours; In the subsequent cultivation process of step 4, the cells are changed every 48-72 hours.
  • the present disclosure also provides a culture medium, including a basal medium and interleukin-2, interleukin-15, and vitamin C added to the basal medium.
  • the basal medium is RPMI-1640 medium.
  • the concentration of interleukin-2 is 1-1000 ng/ml
  • the concentration of interleukin-15 is 1-1000 ng/ml
  • the concentration of vitamin C is 10 ⁇ M-800 mM.
  • the present disclosure also provides a first culture medium, including a second culture medium and a phosphonic acid compound.
  • the concentration of the phosphonic acid compound in the first medium is 1-1000 ⁇ M.
  • the proliferation efficiency of human V ⁇ 9V ⁇ 2T cells is faster and the cell purity is higher, and cultured (from step 3) 10 At -12 days, 90% cell purity can be achieved, and the cell expansion ratio is more than 1000 times (that is, it can expand 100,000 individual V ⁇ 9V ⁇ 2 T cells to at least 100 million individual V ⁇ 9V ⁇ 2 T cells), significantly shortening human V ⁇ 9V ⁇ 2 T cells Expansion culture cycle;
  • the human V ⁇ 9V ⁇ 2 T cells obtained by the expansion of the present disclosure have stronger killing and inhibiting ability on tumors;
  • the human V ⁇ 9V ⁇ 2 T cells obtained by the expansion of the present disclosure have stronger anti-apoptosis ability and longer survival time, and after the cell expansion culture cycle (10-12 days from step 3) ends, the present disclosure expands The increased human V ⁇ 9V ⁇ 2 T cells can continue to survive for about 20 days.
  • Fig. 1 is a comparison diagram of the proliferation efficiency of human V ⁇ 9V ⁇ 2 T cells expanded and cultured using four different culture media;
  • Figure 2 is a comparison chart of the apoptosis rate of human V ⁇ 9V ⁇ 2T cells obtained by using four different culture media to expand and culture human V ⁇ 9V ⁇ 2 T cells;
  • Figure 3 is a comparison chart of the expression levels of NKG2D, a key killer molecule of human V ⁇ 9V ⁇ 2T cells, obtained by expanding and culturing human V ⁇ 9V ⁇ 2 T cells using four different culture media;
  • 4A is a schematic diagram of the tumor size of human V ⁇ 9V ⁇ 2 T cells obtained by traditional amplification method (IL2) after cell treatment of humanized lung cancer mice;
  • 4B is a schematic diagram of the tumor size of human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method (IL2+IL15+VC) of the present disclosure after cell treatment of humanized lung cancer mice;
  • 4C is a schematic diagram of the tumor size of humanized lung cancer mice without treatment
  • 5 is a schematic diagram of the tumor volume change of humanized lung cancer mice under different treatment conditions
  • Figure 6 is a schematic diagram of the survival rate of lung cancer humanized mice under different treatment conditions.
  • basal medium refers to a solution containing nutrients that nourish the growth of mammalian cells.
  • the basic medium provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as vitamins, glucose, buffer systems and essential amino acids.
  • the basal medium is RPMI-1640 medium, D-MEM, MEM, RPMI, Opti-MEM, or a combination thereof.
  • compositions, step, method, article, or device containing the listed elements need not be limited to those elements, but may include other elements not specifically listed or inherent to such a composition, step, method, article, or device Elements.
  • Parts by mass refers to the basic unit of measurement that represents the mass ratio relationship of multiple components.
  • One part can represent any unit mass, such as 1g or 2.689g. If we say that the mass part of component A is a part and the mass part of component B is b part, it means the ratio of the mass of component A to the mass of component a: b. Or, the mass of the A component is aK, and the mass of the B component is bK (K is an arbitrary number, indicating a multiple factor). Not to be misunderstood is that, unlike the number of parts by mass, the sum of the parts by mass of all components is not limited to 100 parts.
  • a and/or B includes (A and B) and (A or B);
  • vitamin C derivative is generally a type of compound that stabilizes the reduced vitamin C enediol structure by introducing other groups at the second carbon atom of vitamin C.
  • examples of vitamin C derivatives include vitamin C phosphates, such as vitamin C phosphate magnesium and vitamin C sodium phosphate; vitamin C palmitate, vitamin C ethyl ether; vitamin C carbohydrates, such as vitamin C glucoside, etc. Wait.
  • Peripheral blood mononuclear cells refer to mononuclear cells isolated from peripheral blood and are commonly used for anti-cancer immunotherapy. Peripheral blood mononuclear cells can be obtained from collected human blood using, for example, Ficoll-Hypaque density gradient method.
  • Peripheral blood mononuclear cells can be obtained from normal persons, subjects or patients at risk of disease.
  • the peripheral blood mononuclear cells used here need not necessarily be derived from autologous cells, and allogeneic peripheral blood mononuclear cells can also be used.
  • malignant disease is used herein in its broadest sense and refers to a disease characterized by uncontrolled cell growth. It includes but is not limited to adrenal cortical cancer, anal cancer, bladder cancer, ependymoma, medulloblastoma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, eye cancer , Gallbladder cancer, gastric cancer, germ cell tumors, extragonads, head and neck cancer, hypopharyngeal cancer, islet cell cancer, laryngeal cancer, leukemia, acute lymphoblastic leukemia, oral cancer, liver cancer, lung cancer and so on.
  • autoimmune disease refers to diseases and conditions caused by the body's immune response against its own tissues, causing prolonged inflammation and subsequent tissue destruction.
  • autoimmune diseases include, but are not limited to, alopecia areata, type 1 diabetes, Guillain-Barré syndrome, multiple sclerosis, rheumatoid arthritis, scleroderma, polymyositis, vitiligo, and systemic lupus erythematosus.
  • infectious disease can be the result of any pathogen. Examples include but are not limited to the results of viral infections such as AIDS, hepatitis B and C, cellular infections, bacterial infections, parasitic and fungal infections.
  • the present disclosure first provides a method for expanding human V ⁇ 9V ⁇ 2T cells, including:
  • Step 1 Extract peripheral blood mononuclear cells (PBMC) from human peripheral blood;
  • PBMC peripheral blood mononuclear cells
  • Step 2 Provide a first culture medium, and use the first culture medium to resuspend the peripheral blood mononuclear cells to obtain a cell suspension;
  • the first medium includes a second medium and a phosphonic acid compound; the second medium includes a basal medium and interleukin-2 (IL-2) and interleukin-15 (added to the basal medium IL-15) and Vitamin C (VitaminC);
  • IL-2 interleukin-2
  • IL-15 interleukin-15
  • Vitamin C Vitamin C
  • Step 3 Inoculate the cell suspension into a cell culture vessel and incubate for 60-100 hours;
  • Step 4 The second culture medium is used for liquid exchange, and subsequent culture processes are all performed by using the second culture medium.
  • the basic medium is RPMI-1640 medium.
  • RPMI is the abbreviation of Roswell Park Memorial Institute, which refers to the Losway Park Memorial Institute.
  • RPMI is a type of cell culture medium developed by the institute, and 1640 is the medium code. When this medium is used, 10% fetal bovine serum is added.
  • the phosphonic acid compounds include zoledronic acid (Zoledronate, ZOL), etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minophosphonic acid.
  • ZOL zoledronic acid
  • etidronic acid etidronic acid
  • ibandronic acid pamidronic acid
  • alendronic acid alendronic acid
  • risedronic acid minophosphonic acid
  • the phosphonic acid compound is zoledronic acid.
  • the cell density of the cell suspension is 3-4 ⁇ 10 6 cells/ml.
  • steps 2 and 3 are to selectively culture the peripheral blood mononuclear cells by culturing the peripheral blood mononuclear cells using a first medium containing phosphonic acid compounds V ⁇ 9V ⁇ 2T cells expand and inhibit the growth of other cells, causing other cells to apoptosis.
  • the cell culture container is a 24-well plate, a 48-well plate or a 96-well plate.
  • step 4 The purpose of the step 4 is to further expand the V ⁇ 9V ⁇ 2T cells with higher purity selectively expanded in the step 3, so as to increase the number of V ⁇ 9V ⁇ 2T cells.
  • the concentration of the phosphonic acid compound in the first medium is 1-1000 ⁇ M
  • the concentration of interleukin-2 is 1-1000 ng/ml
  • the concentration of interleukin-15 is 1-1000 ng/ml
  • the concentration of vitamin C is 10 ⁇ M-800 mM.
  • the cell suspension is inoculated into a cell culture container and cultured for 72 hours.
  • the cells are changed every 48-72 hours.
  • the total culturing time of steps 3 and 4 is usually 10-12 days. At this time point, the number of cells is sufficient and the killing ability is strongest, which is especially suitable for cell therapy.
  • the present disclosure provides a T cell expansion method, including:
  • Step A cultivating a composition containing T cells using a first medium including interleukin-2 and a phosphonic acid compound, and optionally interleukin-15 and vitamin C or vitamin C derivatives to stimulate the T cells;
  • step B the stimulated T cells are cultured using a second medium including interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the present disclosure provides a method for expanding V ⁇ 9V ⁇ 2T cells, including:
  • Step A cultivating a composition containing T cells using a medium including interleukin-2 and a phosphonic acid compound, optionally including interleukin-15 and vitamin C or vitamin C derivatives to stimulate the T cells;
  • Step B Select and isolate V ⁇ 9V ⁇ 2T cells from stimulated peripheral blood mononuclear cells
  • step C the selected and isolated V ⁇ 9V ⁇ 2T cells are cultured using a second medium including interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the present disclosure provides a method for expanding V ⁇ 9V ⁇ 2T cells, including:
  • Step A Extract peripheral blood mononuclear cells (PBMC) from peripheral blood;
  • PBMC peripheral blood mononuclear cells
  • Step B using a medium capable of stimulating T cells to culture the composition containing T cells to stimulate the T cells;
  • Step C Select and isolate V ⁇ 9V ⁇ 2T cells from stimulated peripheral blood mononuclear cells
  • step D the selected and isolated V ⁇ 9V ⁇ 2T cells are cultured using a second medium including interleukin-2, interleukin-15, and vitamin C or vitamin C derivatives.
  • the present disclosure provides a method for inhibiting, preventing or treating infectious diseases, autoimmune diseases or malignant diseases, including:
  • the first subject and the second subject may be the same subject.
  • the first subject and the second subject are different subjects.
  • the peripheral blood mononuclear cells are used as the T cell-containing composition, preferably the malignant disease is cancer, such as lung cancer.
  • the key technical feature of the present disclosure is the use of interleukin-15 and vitamin C in the process of cell culture.
  • interleukin-15 and vitamin C in the expansion culture of human V ⁇ 9V ⁇ 2T cells, it is different from the traditional expansion method (Class 2 (Amplification technology) can improve the proliferation efficiency and cell purity of human V ⁇ 9V ⁇ 2 T cells, the cultured human V ⁇ 9V ⁇ 2T cells have stronger anti-apoptosis ability, longer cell survival time, and higher expression level of key killer molecule NKG2D , So that the ability to kill tumor cells is stronger.
  • the present disclosure solves the problems such as low amplification efficiency and low purity, the problem that the obtained cells have a weak killing function, and the obtained cells Technical problems such as insufficient survival time, aging, and apoptosis.
  • the beneficial effects of the disclosed human V ⁇ 9V ⁇ 2T cell expansion method include:
  • the proliferation efficiency of human V ⁇ 9V ⁇ 2T cells is faster and the cell purity is higher, and cultured (from step 3) 10 At -12 days, 90% cell purity can be achieved, and the cell expansion ratio is more than 1000 times (that is, it can expand 100,000 individual V ⁇ 9V ⁇ 2 T cells to at least 100 million individual V ⁇ 9V ⁇ 2 T cells), significantly shortening human V ⁇ 9V ⁇ 2 T cells Expansion culture cycle;
  • the human V ⁇ 9V ⁇ 2 T cells obtained by the expansion of the present disclosure have stronger killing and inhibiting ability on tumors;
  • the human V ⁇ 9V ⁇ 2 T cells obtained by the expansion of the present disclosure have stronger anti-apoptosis ability and longer survival time, and after the cell expansion culture cycle (10-12 days from step 3) ends, the present disclosure expands The increased human V ⁇ 9V ⁇ 2 T cells can continue to survive for about 20 days.
  • the present disclosure also provides a human V ⁇ 9V ⁇ 2T cell culture medium (also referred to herein as a second culture medium), including a basal medium and interleukin-2 added to the basal medium, Interleukin-15 and vitamin C.
  • the basal medium is RPMI-1640 medium.
  • the concentration of interleukin-2 in the first medium and the second medium is 1-1000ng/ml, or 1-500ng/ml, or 1-200ng/ml, or 70-130ng /ml.
  • the concentration of interleukin-15 in the second medium is 1-1000 ng/ml, or 1-500 ng/ml, or 1-200 ng/ml, or 70-130 ng/ml.
  • the concentration of vitamin C in the second medium is 10 ⁇ M-800 mM, or 20 ⁇ M-400 mM, or 50 ⁇ M-100 ⁇ M.
  • the present disclosure also provides a human V ⁇ 9V ⁇ 2T cell stimulation expansion medium (also referred to herein as a first medium), including the second medium as described above and a phosphonic acid compound.
  • a human V ⁇ 9V ⁇ 2T cell stimulation expansion medium also referred to herein as a first medium
  • a phosphonic acid compound including the second medium as described above and a phosphonic acid compound.
  • the concentration of the phosphonic acid compound in the first medium is 1-1000 ⁇ M.
  • Example 1 Expansion effect of V ⁇ 9V ⁇ 2T cells and characteristics of expanded V ⁇ 9V ⁇ 2T cells
  • the cell density was adjusted to 3-4 ⁇ 10 6 cells/mL with 10% FBS RPMI-1640 complete medium to inoculate the 24-well plate, and 40ng/mL IL- 2 and 50 ⁇ M zoledronic acid stimulation for 3 days.
  • Figure 1 is a comparison diagram of the proliferation efficiency of human V ⁇ 9V ⁇ 2 T cells by using four different cell culture media to stimulate with zoledronic acid.
  • IL2 indicates that the medium obtained by adding IL2 on the basis of the basic medium is used in the entire expansion culture process
  • IL2+VC means that the medium obtained after adding IL2+VC on the basis of the basic medium is used in the entire expansion culture process;
  • IL2+IL15 means that the medium obtained after adding IL2+IL15 on the basis of the basic medium is used in the entire expansion culture process;
  • IL2+IL15+VC means that the medium obtained after adding IL2+IL15+VC on the basis of the basic medium is used in the entire expansion culture process;
  • Figure 2 is a comparison of the apoptosis rate of human V ⁇ 9V ⁇ 2 T cells obtained by expanding and cultivating human V ⁇ 9V ⁇ 2 T cells with four different culture media.
  • IL2, IL2+VC, IL2+IL15, IL2+IL15+ in Figure 2 VC means the same as in Fig. 1.
  • Fig. 3 is a comparison diagram of the expression levels of key killer molecules NKG2D of human V ⁇ 9V ⁇ 2 T cells obtained by expanding and culturing human V ⁇ 9V ⁇ 2 T cells using four different culture media.
  • IL2, IL2+VC, IL2+IL15, IL2 in Fig. +IL15+VC means the same as Figure 1 and Figure 2.
  • the combination of IL-15 and vitamin C can maintain the high-level expression of NKG2D, a key killer molecule of human V ⁇ 9V ⁇ 2 T cells, and maintain high-level expression after 21 days of culture.
  • the combination of IL-15 and VC (that is, the technical solution of the present disclosure) can maintain the high killing capacity of human V ⁇ 9V ⁇ 2 T cells for a longer period of time.
  • mice were purchased from Taconic, SPF grade. Immune-deficient mice use independently ventilated IVC cages. Five mice were housed in each cage. Each isolator is independent HEPA inlet and outlet air, with 24-hour temperature and humidity control, its feeding and litter supply are vacuum packed and ⁇ -radiation sterilized, and sterilized water is used as animal drinking water in this environment Animal packaging is also carried out in a sterile environment. Animals are transported in biologically safe transport boxes to ensure that the breeding and transportation are maintained in the same microbial state to ensure and maintain animal quality. Animal testing was approved by the Experimental Animal Ethics Committee.
  • PBMC PBMC
  • huPBMCs humanized mouse models
  • mice 4-6 weeks old normal Rag2 -/- ⁇ c -/- mice were selected, and after sublethal dose of 300 cGay radiation treatment, 30 ⁇ 10 6 huPBMCs were injected intraperitoneally. After 4 weeks, the surviving humanized mice can be used in the next lung cancer model construction.
  • A549 cells were digested, a cell suspension was prepared, and the concentration of A549 cells was adjusted to 1 ⁇ 10 7 cells/mL with Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • IL-2, IL-2+Vc, IL-2+IL- were injected into the humanized mouse model of lung cancer through the mouse tail vein every 3 days 15.
  • the PBS injection group is the treated control group.
  • IL-2, IL-2+Vc, IL-2+IL-15, IL-2+IL-15+Vc cultured ⁇ T cells were passed as described in the section “In vitro expansion of V ⁇ 9V ⁇ 2 T cells” in Example 1. The method and steps were obtained, and the meanings expressed by IL2, IL2+VC, IL2+IL15, IL2+IL15+Vc are the same as those in FIG. 1 in Example 1.
  • Peripheral blood was drawn at the end of the reinfusion course to detect the percentage of ⁇ T cells and cell activity in the mouse. After the reinfusion, the percentage of ⁇ T cells and the viability of the peripheral blood were drawn every two weeks: NKG2D, PD1, CD107a, Fas , FasL.
  • mice Before killing the mice, detect the colonization of ⁇ T cells in the mice. Simultaneously detect the GFP signal and observe the number and percentage of A549 tumor cells in peripheral blood.
  • FIG. 4A is a schematic diagram of the tumor size of human V ⁇ 9V ⁇ 2 T cells obtained by traditional amplification method (IL2) after cell treatment of humanized lung cancer mice.
  • IL2 traditional amplification method
  • the traditional amplification method (that is, the second type of amplification technique) is to add IL2 to the basal medium for cultivation at all times, and add ZOL in the early stage of the cultivation to stimulate the amplification.
  • FIG. 4B is a schematic diagram of the tumor size of human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method (IL2+IL15+VC) of the present disclosure after cell treatment of humanized lung cancer mice.
  • FIG. 4C is a schematic diagram of tumor size of humanized lung cancer mice without treatment.
  • the humanized lung cancer mice used in the experiments of FIGS. 4A, 4B, and 4C are mice of the same tumor type and basically the same tumor volume, and the tumors shown in FIGS. 4A, 4B, and 4C have the same growth time.
  • the human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method of the present disclosure after cell treatment of humanized lung cancer mice, proved to be the same as those obtained by traditional amplification methods.
  • the expanded human V ⁇ 9V ⁇ 2 T cells of the present disclosure can more effectively inhibit the growth of lung cancer cells (the tumor volume is significantly reduced), and can significantly improve the survival rate of humanized lung cancer mice.
  • humanized lung cancer humanized mice and human lung cancer untreated humanized mice obtained by traditional amplification methods using human V ⁇ 9V ⁇ 2 T cells for cell therapy have all died, and the amplification method of the present disclosure is used All humanized lung cancer humanized mice with human V ⁇ 9V ⁇ 2T cells for cell therapy survived, with a survival rate of 100%, and the tumor of one of the mice completely disappeared (shown in the box of FIG. 4B).
  • Figure 5 is a schematic diagram of the tumor volume changes of lung cancer humanized mice under different treatment conditions.
  • Figure 5 shows the use of traditional amplification methods (IL2) to obtain human V ⁇ 9V ⁇ 2 T cells on lung cancer humanized mice.
  • IL2+IL15+VC traditional amplification methods
  • human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method (IL2+IL15+VC) of the present disclosure are used for cell therapy of lung cancer humanized mice and tumor volume changes of untreated lung cancer humanized mice.
  • IL2+IL15+VC the tumor volume of humanized lung cancer humanized mice treated with human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method of the present disclosure
  • Figure 6 is a schematic diagram of the survival rate of lung cancer humanized mice under different treatment conditions.
  • Figure 6 shows the use of traditional amplification methods (IL2) to obtain human V ⁇ 9V ⁇ 2 T cells on lung cancer humanized mice.
  • IL2+IL15+VC human V ⁇ 9V ⁇ 2 T cells obtained by the amplification method of the present disclosure
  • This Example 1 provides a second culture medium, including RPMI-1640 medium and interleukin-2, interleukin-15 and vitamin C added to the RPMI-1640 medium.
  • the concentration of interleukin-2 is 300 ng/ml
  • the concentration of interleukin-15 is 500 ng/ml
  • the concentration of vitamin C is 100 mM.
  • This example 2 provides a second culture medium, including RPMI-1640 medium and interleukin-2, interleukin-15 and vitamin C added to the RPMI-1640 medium.
  • the concentration of interleukin-2 is 500 ng/ml
  • the concentration of interleukin-15 is 700 ng/ml
  • the concentration of vitamin C is 300 mM.
  • This embodiment 3 provides a first culture medium, including the second culture medium as described in embodiment 1 and zoledronic acid.
  • the concentration of zoledronic acid in the first medium is 300 ⁇ M.
  • This Example 4 provides a first medium, including the second medium as described in Example 2 and zoledronic acid.
  • the concentration of zoledronic acid in the first medium is 500 ⁇ M.
  • the present disclosure illustrates the detailed process equipment and process flow of the present disclosure through the above embodiments, but the present disclosure is not limited to the detailed process equipment and process flow described above, which does not mean that the present disclosure must rely on the above detailed process equipment and process flow The process can only be implemented. Those skilled in the art should understand that any improvement to the present disclosure, equivalent replacement of various raw materials of the disclosed product, addition of auxiliary components, selection of specific methods, etc., fall within the protection scope of the present disclosure.
  • the proliferation efficiency of human V ⁇ 9V ⁇ 2 T cells is faster and the cell purity is higher, and the amplification method of the present disclosure significantly shortens the human V ⁇ 9V ⁇ 2 T cell expansion culture cycle.
  • the human V ⁇ 9V ⁇ 2 T cells amplified by the present disclosure have stronger killing and inhibiting ability on tumors.
  • the human V ⁇ 9V ⁇ 2 T cells amplified by the present disclosure have stronger anti-apoptotic ability and longer survival time.

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